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Directed evolution of P-glycoprotein cysteines reveals site-specific, non-conservative substitutions that preserve multidrug resistance.

ABSTRACT: Pgp (P-glycoprotein) is a prototype ABC (ATP-binding-cassette) transporter involved in multidrug resistance of cancer. We used directed evolution to replace six cytoplasmic Cys (cysteine) residues in Pgp with all 20 standard amino acids and selected for active mutants. From a pool of 75000 transformants for each block of three Cys, we identified multiple mutants that preserved drug resistance and yeast mating activity. The most frequent substitutions were glycine and serine for Cys427 (24 and 20%, respectively) and Cys1070 (37 and 25%) of the Walker A motifs in the NBDs (nucleotide-binding domains), Cys1223 in NBD2 (25 and 8%) and Cys638 in the linker region (24 and 16%), whereas close-by Cys669 tolerated glycine (16%) and alanine (14%), but not serine (absent). Cys1121 in NBD2 showed a clear preference for positively charged arginine (38%) suggesting a salt bridge with Glu269 in the ICL2 (intracellular loop 2) may stabilize domain interactions. In contrast, three Cys residues in transmembrane ?-helices could be successfully replaced by alanine. The resulting CL (Cys-less) Pgp was fully active in yeast cells, and purified proteins displayed drug-stimulated ATPase activities indistinguishable from WT (wild-type) Pgp. Overall, directed evolution identified site-specific, non-conservative Cys substitutions that allowed building of a robust CL Pgp, an invaluable new tool for future functional and structural studies, and that may guide the construction of other CL proteins where alanine and serine have proven unsuccessful.

SUBMITTER: Swartz DJ

PROVIDER: S-EPMC4069687 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Directed evolution of P-glycoprotein cysteines reveals site-specific, non-conservative substitutions that preserve multidrug resistance.

Swartz Douglas J DJ Mok Leo L Botta Sri K SK Singh Anukriti A Altenberg Guillermo A GA Urbatsch Ina L IL

Bioscience reports 20140625 3

Pgp (P-glycoprotein) is a prototype ABC (ATP-binding-cassette) transporter involved in multidrug resistance of cancer. We used directed evolution to replace six cytoplasmic Cys (cysteine) residues in Pgp with all 20 standard amino acids and selected for active mutants. From a pool of 75000 transformants for each block of three Cys, we identified multiple mutants that preserved drug resistance and yeast mating activity. The most frequent substitutions were glycine and serine for Cys427 (24 and 20 ...[more]

PMID: 24825346

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Project description:ATP-binding cassette (ABC)-transporters protect tissues by pumping their substrates out of the cells in many physiological barriers, such as the blood-brain barrier, intestine, liver, and kidney. These substrates include various endogenous metabolites, but, in addition, ABC transporters recognize a wide range of compounds, therefore affecting the disposition and elimination of clinically used drugs and their metabolites. Although numerous ABC-transporter inhibitors are known, the underlying mechanism of inhibition is not well characterized. The aim of this study is to deepen our understanding of transporter inhibition by studying the molecular basis of ligand recognition. In the current work, we compared the effect of 44 compounds on the active transport mediated by three ABC transporters: breast cancer resistance protein (BCRP and ABCG2), multidrug-resistance associated protein (MRP2 and ABCC2), and P-glycoprotein (P-gp and ABCB1). Eight compounds were strong inhibitors of all three transporters, while the activity of 36 compounds was transporter-specific. Of the tested compounds, 39, 25, and 11 were considered as strong inhibitors, while 1, 4, and 11 compounds were inactive against BCRP, MRP2, and P-gp, respectively. In addition, six transport-enhancing stimulators were observed for P-gp. In order to understand the observed selectivity, we compared the surface properties of binding cavities in the transporters and performed structure-activity analysis and computational docking of the compounds to known binding sites in the transmembrane domains and nucleotide-binding domains. Based on the results, the studied compounds are more likely to interact with the transmembrane domain than the nucleotide-binding domain. Additionally, the surface properties of the substrate binding site in the transmembrane domains of the three transporters were in line with the observed selectivity. Because of the high activity toward BCRP, we lacked the dynamic range needed to draw conclusions on favorable interactions; however, we identified amino acids in both P-gp and MRP2 that appear to be important for ligand recognition.

Dataset Information

Directed evolution of P-glycoprotein cysteines reveals site-specific, non-conservative substitutions that preserve multidrug resistance.

Publications

Directed evolution of P-glycoprotein cysteines reveals site-specific, non-conservative substitutions that preserve multidrug resistance.

Similar Datasets

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